This project involves the study of a rapidly emerging group of immune receptors that exist in both activating and inhibitory configurations. Some of these receptors associate with the novel signal transduction chain Dap12. Our studies focus on the biochemistry of the Dap12 signal transduction pathway and elucidation and characterization of Dap12-coupled receptor families.Our studies currently largely involve the study of the Ly49s of NK cells and the Triggering Receptors Expressed on Myeloid cells (TREM). In NK cells we continue to focus on dissection of the role of a variety of complex signaling adaptors in the transmission of the Dap12 signal. Our model systems includes NK cell line expressing Dap12-coupled receptors and mice with targeted mutations in any of several signal transduction proteins. The TREM are a rapidly emerging, Dap12-coupled immune receptor family. TREMs are involved in the amplification of septic shock, and the maturation of dendritic cells and osteoclasts. Despite the established role of TREMs in both innate and adaptive immunity the signal transduction of the family is still relatively unknown. Using a newly developed chimeric receptor approach, we are defining the involvement of a variety of proteins in the TREM/Dap12 signaling pathway in the myeloid compartment. Consequently, analysis of the DC and macrophages of our null mice is ongoing.In addition to the activating members of the TREM family, we recently described TREM Like Transcript-1 (TLT-1), a putative inhibitory receptor within the TREM cluster that is expressed exclusively in the alpha granules of megakaryocytes and platelets. Our BAC-derived gene targeting of TLT-1 complete and analysis is underway. The BAC technology allowed the rapid modification of large pieces of DNA using in vivo recombination in bacteria, and the expedited targeting of TLT-1 in C57BL/6 ES cells. Therefore, the analysis of these mice will not be affected in anyway by differences in genetic backgroundAs we develop the mouse model we continue to dissect the intracellular binding partners of TLT-1. For these studies a variety of approaches are being used including GST-fusion proteins and co-immunoprecipitation studies using megakayrocytes.